The study of lysine methylation is an emerging field; tremendous progress has been made in understanding this post-translational modification (PTM) on histones and its roles in epigenetic regulation. Many of the enzymes that methylate or demethylate histones have been identified in the recent years, and deregulation of several of these enzymes has been linked to tumorgenesis. One trend that emerges from the study of PTMs on histones is that many of the enzymes that modify histones can also modify and regulate the function of nonhistone proteins. Our understanding of lysine methylation on nonhistone proteins is almost non-existent since methylation sites on three eukaryotic proteins have been identified so far. The goals of this proposal are to develop the necessary tools that will enable the study of lysine methylation and to conduct the first survey of the human methyllysine proteome. We propose strategies to produce pan specific antibodies to the mono-, di-, and trimethyllysine. The generation of pan specific antibodies to the three forms of methyllysine will enable scientists to easily study lysine methylation on any protein. Our strategy to identify methyllysine-containing proteins from HeLa cells is novel in that it incorporates a simple prefractionation of the cell extracts to reduce the complexity of the sample and therefore increase our likelihood to immunoaffinity purify the methyllysine peptides with the pan specific antibodies. The experiments outlined in this proposal will greatly advance our understanding of the biology of lysine methylation in signal transduction pathways. We envision that pan specific antibodies and the catalog of methyllysine proteins in the human proteome will be foundation for important breakthroughs in this untapped area of research. Given that deregulation of lysine methylation enzymes have been linked to various human diseases, including cancer, we believe that the tools and discoveries describe in this proposal will lead to future scientific breakthroughs that will have a direct impact on human health. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: Deregulation of lysine methylation has been linked to various human diseases, including cancer. This proposal outlines the development of much-needed reagents and methods for the study of lysine methylation. The successful completion of this proposed project will lead the development of important tools for the study of lysine methylation and build the foundation for future scientific discoveries that will have a direct impact on public health. [unreadable] [unreadable] [unreadable]